DE102022208620A1 - Microscopy method, especially for fluorescence correlation spectroscopy - Google Patents

Abstract

The invention relates to a microscopy method in which a focused beam of excitation radiation (AS) is directed into an examination site (17) of an object (7) to be examined, an excitation volume being generated in the object (7). At least one structure (15) of the object (7) is identified using an overview image and at least one of the identified structures (15) is defined as a reference structure (16). A spatial relationship is established between the positions of the examination site (17) and the reference structure (16). Detection radiation (DS) coming from the excitation volume is recorded as measured values over a total measurement period, with the total measurement period being divided into several measurement intervals. Before at least every second measurement interval, the current position of the focused beam of rays is compared with a current position of the examination site (17). If there is an unacceptable deviation in the current positions, the positioning of the focused beam of rays is corrected. The invention also relates to a microscope (M) for carrying out the procedure.

Description

The invention relates to a microscopy method that can be used in particular in the field of fluorescence correlation spectroscopy.

In the field of confocal scanning microscopy, especially confocal laser scanning microscopy, fluorescence correlation spectroscopy (FCS) is a proven method, for example, to investigate the dynamics of the behavior of molecules in cells.

For example, using a laser scanning microscope (LSM), the diffusion of a fluorescently labeled molecule or a self-fluorescent molecule into and out of the confocal volume of the LSM can be determined. A confocal volume, also referred to below as an excitation volume, is created in cooperation with a so-called pinhole by focusing a beam of excitation radiation and directing it into an object to be examined. The excitation volume has an extent around the optical axis along which the excitation radiation is irradiated (illumination axis). In addition, the excitation volume is extended a certain distance along the optical axis (Z direction; direction of the z axis).

In fluorescence correlation spectroscopy, the movements and optical behavior of fluorescent molecules that are in the excitation volume or that pass through it are recorded and evaluated. The position of the excitation volume (examination location) is kept constant relative to the sample over the entire period of an FCS measurement.

However, typical FCS measurements take several seconds, occasionally up to minutes. Since FCS measurements are normally carried out on living samples, the sample, or the structures in the sample to be examined, can move during the FCS measurement and thus migrate out of the excitation volume. The observed movement of a fluorescent molecule is then not only caused by an actual diffusion movement of this molecule, but is also distorted by an unintentional displacement of the sample. As a result of such a shift, the position of the excitation volume has changed undesirably relative to the sample. In certain configurations of the FCS process, such shifts can be very disruptive. For example, FCS measurement values in the so-called Airyscan FCS are potentially strongly influenced if, for example, a membrane is to be measured exactly on a surface or in a channel ( L. Scipioni, L. Lanzanó, A. Diaspro & E. Gratton, 2018; “Comprehensive correlation analysis for superresolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector,” NATURE COMMUNICATIONS 9: 5120 ).

The object of the invention is to provide a possibility by means of which an effective correction of occurring shifts between excitation radiation and a desired examination location takes place.

The task is solved with the subject matter of the independent claims. Advantageous further training is the subject of the dependent claims.

The problem is solved using a microscopy method in which a focused beam of excitation radiation is directed into an examination site of an object to be examined, with an excitation volume being generated in the object. The examination location is determined by the position of the excitation volume in the object. To simplify, it can be assumed that if the excitation radiation is directed essentially perpendicularly onto the object, the point of impact of the excitation radiation on the object can be viewed as the examination site.

The object is in particular a biological sample, for example a cell or a tissue. The object can also be a sol, a gel, a suspension, a solution or a body that is sufficiently transparent for the excitation radiation and the fluorescence radiation.

At least one structure of the object is identified based on at least one overview image captured along an image capture axis. At least one of the identified structures is set as a reference structure. The overview image preferably extends orthogonally to the image capture axis. In further embodiments of the invention, the overview image can extend at an angle other than 90° to the image capture axis, in particular if the image capture axis is directed at the object at an acute angle. The overview image can be captured with a camera that is present in addition to a detection lens or a common illumination and detection lens. In further embodiments of the invention, the overview image can be captured using the detector, which is also used to capture the measured values, in particular FCS measured values (see below). The overview image can be created using the detector scanning microscope, in particular by means of the detector of a laser scanning microscope (LSM). In the latter embodiment, the image capture axis coincides with an optical axis of the lens used for detection. Another possible embodiment of the invention uses the detection beam path of the scanning microscope, but uses a further detector to capture an overview image. For this purpose, the detected radiation can optionally be directed to the further detector for the purpose of capturing the overview image.

The at least one reference structure is, for example, a section of a cell membrane or cell wall, a holding and supporting structure such as collagen fibers or a section of cell organelles such as the endoplasmic reticulum, mitochondria, chloroplasts or vacuoles. Preferably, such structures are selected as reference structures in which no or only negligible changes in their position with respect to the examination site are to be expected during the desired total measurement duration of the method. For example, a reference point is selected and defined by the reference structure. It is also possible to determine a geometric center of gravity of the reference structure. In order to improve the accuracy of position control, multiple reference points and/or centers of gravity of different reference structures can be defined. Such an approach allows the plausibility of several results of a spatial relationship (see below) to be checked with one another. In addition, it is then possible to only take into account those reference points or focal points that can be clearly determined or whose results are plausible. A clear determination of the reference point can be made more difficult or prevented, for example, as a result of an unfavorable relative position of the reference structure during the capture of one of the overview images.

The examination location can be determined using the overview image. It is also possible to select the examination site and only then search for a suitable reference structure in the area surrounding the examination site.

The examination location can be determined manually by a user of the method according to the invention. However, this is advantageously carried out using known image evaluation methods and/or using image evaluation methods based on machine learning and/or artificial intelligence.

The image capture axis and an illumination axis are advantageously aligned parallel to one another and advantageously have no or only a small lateral offset from one another. In this way, less computing power is required to correlate the information collected along each two axes.

A spatial relationship is established between the position of the examination site and the reference point or center of gravity of the reference structure. For the sake of simplicity, reference is made below to a reference structure. Such a relationship can be defined and saved, for example, in the form of a calculation rule or a specification of positions in a coordinate system and their deviation from one another (distances and directions; vectors). With this step of the method, a target position of the examination location can be determined, in particular calculated, at any time based on a current position of the reference structure.

Accordingly, multiple spatial relationships are established when multiple reference structures are selected. The method may include a plausibility check of the results of the spatial relationship calculations. This can be used, for example, when taking into account several reference structures, to check whether the results are consistent or, for example, whether an individual result deviates from the other results by more than permissible. In such a case, the strongly different result can, for example, be rejected.

If molecules that can be stimulated to emit fluorescent radiation are present within the excitation volume, these are stimulated by the action of the excitation radiation in the excitation volume to emit fluorescent radiation, which is subsequently recorded as detection radiation and stored in the form of measured values.

The total measurement time intended for recording measured values as part of the method according to the invention is divided into several measurement intervals. Measured values are therefore recorded at least in two measurement intervals during the execution of the method.

At least before every second measurement interval, but preferably before each measurement interval, the current position of the focused beam of rays is compared with a current position of the examination site. This is done by determining a current position of the reference structure and using the spatial relationship to determine the current position of the examination location. In this way, a current actual position of the examination location, which is determined by its relative position to the selected reference limit structure is set, can be compared with the current actual position of the focused beam of rays. If an unacceptable deviation between the current positions of the focused beam of rays and the examination site is detected, the positioning of the focused beam of rays is corrected. Well-known algorithms such as cross-correlation can be used to determine the deviations and the necessary correction movements.

The method according to the invention can optionally include further steps.

The core of the invention is a procedure by means of which, on the one hand, measured values can be recorded and evaluated over sufficiently long periods of time, but on the other hand, a regular control and optional correction of the positioning of the focused beam of the excitation radiation, as a result of the excitation volume, can be carried out with regard to the desired examination location can.

In contrast to well-known methods of “single-particle tracking” (e.g. Andrew J. Berglund and Hideo Mabuchi, 2005; “Tracking-FCS: Fluorescence correlation spectroscopy of individual particles”, Optics Express Vol. 13, Issue 20, pp. 8069- 8082; https://doi.org/10.1364/OPEX.13.008069), not a single fluorescent molecule is followed on its path, but at least one reference structure that cannot be measured is used and the beam of the focused excitation radiation is kept constant relative to the reference structure . Any molecules can then diffuse through the excitation volume, which may be excited to emit fluorescent radiation, but cannot be pursued further once they have left the excitation volume.

When carrying out a microscopy method according to the invention, the measured values are recorded over a plurality of measurement intervals, each of the measurement intervals having a duration of at least 0.5 seconds. With a duration of the measurement intervals of at least 0.5 seconds, advantageously one second with a total measurement duration of, for example, ten seconds, an advantageous relationship between the duration of the measured value acquisition and a frequency of the control intervals of the current positioning is achieved. In a further advantageous embodiment of the invention, measuring intervals of ten seconds each are used with a total measuring time of 100 seconds.

In order to carry out the comparison of the current position of the focused beam of rays with the current position of the examination site before each of the measurement intervals, in an advantageous embodiment of the method, at least one overview image of the object is recorded. The overview image advantageously extends orthogonally to the image capture axis. However, it can also be recorded at a detection angle other than 90°, which requires increased calculations to evaluate the overview image. These overview images can optionally cover a smaller object field around the image acquisition axis than the overview image used to identify the structures.

Before the first measurement interval, the overview image that was also used to identify the structures can be used to compare the positions.

The overview images can be wide-field images or images captured using an LSM. Depending on the optical properties of the object, imaging methods such as contrast methods, fluorescence imaging or laser scanning microscopy can be used.

By using an overview image, which extends in particular essentially in an xy plane of a Cartesian coordinate system, a possible deviation in the positions of focused beams of excitation radiation and the examination location can be determined. In order to also enable a correction in the Z direction, in a further advantageous embodiment of the method according to the invention, the comparison of the current position of the focused beam of rays with the current position of the examination site can be carried out using a stack of overview images, the overview images extending orthogonally to the image acquisition axis and are stacked along the image capture axis, i.e. in the direction of the z-axis. To limit the time required for position control, such a stack (Z-stack) can, for example, only have three to five overview images in different levels. Such Z-stacks can also be used to identify and define the structures or the at least one reference structure.

In order to achieve reliable reproducibility of decisions regarding the identification and selection of the structures as well as the determination of the reference structure(s) and/or the selection and determination of the examination location, these process steps can be automated individually or in their entirety. For this purpose, the processes mentioned can be carried out using known image evaluation methods the decisions made can be advantageously tracked and checked using adjustable and documented parameters.

Additionally or alternatively, image evaluation methods based on machine learning and/or artificial intelligence may be used to perform some or all of the identification, selection and determination processes.

Unlike in the prior art, using the method according to the invention, measured values of a complete measuring process are recorded at intervals and the positions are repeatedly checked and, if necessary, corrected. The measured values recorded during the measuring intervals can be evaluated in various ways after the total measuring period of the measuring process has expired. Firstly, it is possible to average or sum up the measured values of the measuring intervals. On the other hand, the measured values of the individual measuring intervals can be strung together to form a common measuring interval and an evaluation can be carried out over the entire duration of the summed up measuring intervals. It is also possible to evaluate the measured values of the individual measuring intervals separately, for example by adapting them to mathematical models (“fitting”). The results determined, in particular the factors and/or terms used to fit the models (“fit parameters”), can then be averaged.

For all evaluation methods, it is possible to make a selection of measurement intervals based on which the evaluation is carried out. All or only some of the measurement intervals can be selected and taken into account.

The microscopy method according to the invention can be used particularly advantageously in the embodiment as a fluorescence correlation spectroscopy method (FCS method).

To carry out the method, a microscope or an optical device can be used, which is used in particular for fluorescence correlation spectroscopy. In addition to the required optical elements and beam paths, such a device has a control unit that is configured to carry out the method according to the invention. The microscope according to the invention also has a scanning device and/or a controlled movable sample table in an excitation beam path for aligning a relative position of the focused beam of rays and an object to be examined.

The control unit can be, for example, a PC, an FPGA, a CPU or a microcontroller. The control unit receives the measured values recorded during the measuring intervals and saves them. It is also configured to identify the structures and optionally select the reference structure, determine the spatial relationship and optionally calculate the current positions. The control unit generates control commands based on the determined positions of the focused beam of rays and the examination location. These are transmitted to drives of at least one scanning device in the excitation beam path and/or a sample table. As a result of the execution of the control commands, the positioning of the beam and examination site is corrected if necessary.

A detector for capturing an overview image of an object to be examined and a detector for detecting fluorescent radiation are arranged in a detection beam path. The fluorescent radiation to be detected is excited by the effect of the excitation radiation in the object and emitted by it.

In further embodiments of the device, an additional camera can be present for capturing the overview image.

• 1 eine schematische Darstellung eines ersten Ausführungsbeispiels eines erfindungsgemäßen Mikroskops;
• 2 ein vergrößerter Bildausschnitt von Objektiv und Kamera gemäß eines zweiten Ausführungsbeispiels des erfindungsgemäßen Mikroskops;
• 3 eine schematisierte Darstellung eines zu untersuchenden Objekts sowie identifizierte Strukturen des Objekts und beispielhafte Lagen eines Untersuchungsorts in einer Draufsicht; und
• 4 ein Ablaufdiagramm einer Ausgestaltung des erfindungsgemäßen Verfahrens.

The invention is explained in more detail below using exemplary embodiments and illustrations. Show it:

• 1 a schematic representation of a first embodiment of a microscope according to the invention;
• 2 an enlarged image detail of the lens and camera according to a second exemplary embodiment of the microscope according to the invention;
• 3 a schematic representation of an object to be examined as well as identified structures of the object and exemplary locations of an examination site in a top view; and
• 4 a flowchart of an embodiment of the method according to the invention.

The illustrations are very simplified and are limited to the technical elements required for explanation. The beam paths are also shown in a very simplified manner.

An exemplary embodiment of a device, in particular a microscope M, comprises a light source 1, for example a laser light source, from which a beam of rays of a type emanates radiation AS is emitted and guided along an excitation beam path 2 ( 1a) . Optional optical elements for shaping and/or collimating the excitation radiation AS are not shown.

The excitation radiation AS impinges on a main color splitter 3, which is transparent to the excitation radiation AS and allows it to pass through. After the main color splitter 3, the excitation radiation AS passes through a section of the beam path of the device, which is referred to as the common beam path 29 and along which the excitation radiation AS and a detection radiation DS (see below) are guided together or can be guided together.

By means of a subsequently arranged scanner 5, the beam of excitation radiation AS, previously deflected by means of a mirror 4, can be deflected in a controlled manner and directed into the entrance pupil EP of an objective 6. The mirror 4 allows a compact design and can be missing in other versions of the device if no deflection of the excitation beam path 2 is required or provided.

The excitation radiation AS, which is deflected in a controlled manner by means of the scanner 5, is focused by the effect of the objective 6 into a sample space in which an object 7 to be examined (sample 7) can be present on a sample table 8. The excitation radiation AS irradiated in such a focused manner causes a confocal excitation volume (not shown) in the sample 7. The sample table 8 is optionally adjustable by means of a drive 13 at least in the xy plane, optionally also in the direction of the Z axis.

A detection radiation DS caused in the sample 7 by the excitation radiation AS in the confocal excitation volume is detected with the objective 6 and along a detection beam path 9 (shown with broken solid lines), which coincides with the excitation beam path 2 up to the main color splitter 3 (common beam path 29), guided.

In further embodiments of the device according to the invention, the detection radiation DS can be detected using a further objective (not shown). In such a case, the excitation beam path 2 and the detection beam path 9 can be completely separated from one another or they can be combined back into the common beam path 29, for example by means of a further color splitter (not shown).

In the exemplary embodiment shown, the detection radiation DS is converted into a stationary beam (“descanned”) as a result of passing through the scanner 5 and reaches the skin color divider 3. This is reflective for the wavelength of the detection radiation DS, which is different from the wavelength of the excitation radiation AS. The detection radiation DS reflected on the main color splitter 3 reaches an optics 10 present in the detection beam path 9. This directs the detection radiation DS onto a detector 12. In further embodiments, the transmittance and reflectivity of the main color splitter 3 can also be reversed, so that the excitation radiation AS is reflected and the detection radiation DS is transmitted. The beam paths 2 and 9 must then be designed accordingly.

In further embodiments, the detector 12 can be preceded by a pinhole (not shown) in the form of a pinhole or slit diaphragm in an intermediate image plane in order to mask out extra-focal components of the detection radiation DS. If the detector 12 is also to be used to capture the overview image, the pinhole is advantageously adjustable in terms of its opening and/or can be moved into the detection beam path 9 or out of the detection beam path 9. In such an embodiment of the invention, the detector 12 can be, for example, a secondary electron multiplier (photon multiplier; PMT). The pinhole can advantageously be adjusted in a controlled manner with regard to its opening width and/or into or out of the detection beam path 9

In a further embodiment, the detector 12 can be an array of a number of individually readable detector elements, as is the case, for example, with an Airy detector (see above) or a SPAD array (Single Photon Avalanche Diode). The detector 12 is then arranged in an intermediate image plane, with the detector elements each functioning as a pinhole. Detector elements can advantageously be optionally connected together in order to set the size of a resulting pinhole. This configuration is particularly suitable for use as an FCS method.

In order to be able to capture and provide the overview images required for the method according to the invention, a camera 14 can be present, the image capture axis BeA of which is at a known angle to the illumination axis BA (corresponds to the optical axis oA of the excitation beam path 2) and is directed towards the sample 7. An inclined arrangement of the camera 14 makes technical sense when there is little available installation space and/or when the camera 14 has large dimensions.

If the camera 14 is dimensioned sufficiently small in relation to the lens 6, the illumination axis BA and the image capture axis BeA can be aligned at a small angle of up to 25 ° or parallel to one another, as is the case in the enlarged image section of the 2 is shown. In further embodiments of the invention, the camera 14 can be dispensed with. The overview image is then captured using the detector 12 and evaluated using a correspondingly configured control unit 11. In such an embodiment, the optical axis oA and the image capture axis BeA coincide. In further embodiments of the device, the camera 14 can also be arranged in the detection beam path 9, with the course of the detection beam path 9 between the detector 12 and the camera 14 being changed in a controlled manner depending on the image to be captured.

The scanner 5, the drive 13, the camera 14 and optionally the light source 1 are connected to the control unit 11 in a connection suitable for exchanging data and control commands. The control unit 11 is, for example, a computer or a suitable control circuit.

Optionally, the control unit 11 and the detector 12 can be connected to one another, for example to enable the control unit 11 to generate control commands based on the detected brightness information of the detector 12 and/or to validate them. These control commands are used, for example, to control the light source 1, the scanner 5 and/or the optional drive 13 of the sample table 8.

The 3 and 4 illustrate how the invention works. Shown in 3 an overview image of a sample 7, for example a cell, along the illumination axis BA (see 1 and 2 ). Due to the different diffraction and refraction behavior of the various components of the sample 7, a number of structures 15 can be identified. Using predetermined criteria, one of the structures 15 is selected as a reference structure 16. From this, a point, for example a prominent point on the reference structure 16, is defined as a reference point to which the angles and distances described below are related. Alternatively, the center of gravity of the reference structure 16 can be determined and used as a reference point.

The user or by means of an algorithm, for example based on the overview image, determines an examination location 17 at which the measurement, in particular an FCS measurement, is to take place. A spatial relationship is determined between the reference structure 16 and the examination site 17, which is given, for example, by an angle α (alpha) and a distance d.

The focused beam of the excitation radiation AS is directed into the examination site 17 and for the duration of a first measurement interval (see 4 ), FCS measurement values are recorded and saved.

After the first measurement interval, the excitation radiation AS is switched off or dimmed and another overview image is captured. This can capture a smaller area of the sample 7 than the overview image used initially. The current positions (actual positions) of the reference structure 16 and the examination location 17 are determined. The current position of the reference structure 16 can be determined from the newly captured overview image. Based on the spatial relationship to the examination site 17, its current position can then be determined. At the same time, the current orientation of the beam of excitation radiation AS is known, for example due to the deflection of the scanner 5 and/or the positioning of the sample table 8. If the point of impact of the excitation radiation AS and the examination site 17 match sufficiently, i.e. their actual positions do not deviate from each other by more than a permissible tolerance level and the actual positions and target positions match, the FCS measurement is continued in the next measurement interval.

On the other hand, if the point of impact of the excitation radiation AS and the examination point 17 deviate from one another in an impermissible manner, the relative position of both is corrected. In 3 An example of an excitation radiation AS is shown (broken solid line), which deviates significantly from the specified examination location 17. By means of the control unit 11, control commands are generated, through the execution of which the scanner 5 and/or the sample table 8 are aligned accordingly so that the excitation radiation AS hits the examination site 17 again.

The control described and, if necessary, the correction of the positions of the examination site 17 and excitation radiation AS is carried out before each further measurement interval until a previously determined number of measurement intervals has been completed or the process is ended by a user.

The duration of the individual measurement intervals corresponds to the total measurement duration of the FCS measurement. For a subsequent evaluation of the recorded FCS measurement values, the measurement intervals can be strung together to form a common measurement interval and evaluated as a temporally coherent sequence. It is also possible, select some of the measurement intervals for the evaluation. In a further embodiment of the method, the FCS measured values of the measurement intervals are averaged and the measurement interval thus obtained is evaluated with the averaged values. Furthermore, measured values of the measurement intervals can be adapted to mathematical models and the results obtained can be further used, for example averaged.

Reference symbols

1

light source

2

excitation beam path

3

Main color divider

4

Mirror

5

scanner

6

lens

7

sample

8th

Sample table

9

Detection beam path

10

optics

11

Control unit

12

detector

13

Drive (of the sample table 8)

14

camera

15

structure

16

Reference structure

17

Examination location

29

common beam path

AS

excitation radiation

B.A

Illumination axis

BeA

Image capture axis

D.S

Detection radiation

d

distance

E.P

Entrance pupil (of lens 6)

M

microscope

oA

optical axis (of the detection beam path 9)

alpha

angle

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Non-patent literature cited

• L. Scipioni, L. Lanzanó, A. Diaspro & E. Gratton, 2018; “Comprehensive correlation analysis for superresolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector,” NATURE COMMUNICATIONS 9: 5120 [0005]

Claims (10)

Microscopy method in which - a focused beam of excitation radiation (AS) is directed into an examination site (17) of an object (7) to be examined, an excitation volume being generated in the object (7); - at least one structure (15) of the object (7) is identified based on an overview image and at least one of the identified structures (15) is defined as a reference structure (16); - a spatial relationship between the positions of the examination site (17) and the reference structure (16) is determined; - detection radiation (DS) coming from the excitation volume is recorded as measured values over a total measurement period; - the total measurement duration is divided into several measurement intervals; - at least before every second measuring interval o the current position of the focused beam of rays is compared with a current position of the examination site (17) by determining a current position of the reference structure (16) and using the spatial relationship to determine the current position of the examination site (17); o if there is an impermissible deviation in the current positions of the focused beam of rays and the examination site (17), the positioning of the focused beam of rays is corrected. microscopy method Claim 1 , characterized in that each of the measurement intervals has a duration of at least 0.5 seconds. microscopy method Claim 1 , characterized in that each of the measurement intervals has a duration of at least one second. Microscopy method according to one of the preceding claims, characterized in that the identification of structures (15) of the object (7) and optionally the determination of the reference structure (16) is carried out automatically by means of an image evaluation method. Microscopy method according to one of the preceding claims, characterized in that the comparison of the current position of the focused beam of rays with the current position of the examination site (17) is carried out using an overview image. Microscopy method according to one of the preceding claims, characterized in that the comparison of the current position of the focused beam of rays with the current position of the examination site (17) is carried out using a stack of overview images, the overview images extending orthogonally to and along an image acquisition axis (BeA). the image acquisition axis (BeA). Microscopy method according to one of the preceding claims, characterized in that an average value of the measured values is calculated via a selection of the measuring intervals of a total measuring period. Microscopy method according to one of the Claims 1 until 6 , characterized in that the measured values of a selection of the measuring intervals of a total measuring duration are combined and evaluated to form a common measuring interval. Microscopy method according to one of the preceding claims, wherein this is designed as a fluorescence correlation spectroscopy method (FCS method), in particular as an Airyscan FCS. Microscope (M) comprising in an excitation beam path (2): a light source (1) for providing excitation radiation (AS); optical elements (6) for generating a focused beam of excitation radiation (AS); a scanning device (5) and/or a controlled movable sample table (8) for aligning a relative position of the focused beam of excitation radiation (AS) and an object (7) to be examined; in a detection beam path (9): either a detector (12) for detecting detection radiation (DS), which is excited by the effect of the excitation radiation (AS) in the object (7) and emitted by it, and for detecting an overview image of the object ( 7); or a detector (12) for detecting detection radiation (DS), which is excited by the effect of the excitation radiation (AS) in the object (7) and emitted by it, and a camera (14) for detecting an overview image of the object (7) ; and a control unit (11) which is used to carry out the method according to one of the Claims 1 until 9 is configured.

Images